Process and composition for amino-terminal, α-aspartyl and α-glutamyl dipeptide esters

ABSTRACT

Reaction of a ketoxime-derivatized resin with a strong acid salt of aspartic anhydride or glutamic anhydride yields a novel aspartyl or glutamyl ketoxime ester-derivatized resin, wherein the aspartyl or glutamyl groups are esterified predominantly at the α-carboxyl group and wherein the aspartyl or glutamyl groups are not covalently protected at the amino group or the carboxyl group that is not esterified. Aminolysis in the presence of a weak acid of the novel aspartyl or glutamyl ketoxime ester-derivatized resin, wherein the aspartyl or glutamyl groups remain as the strong acid salt, with a salt of an amino acid with a base or an amino acid ester yields the corresponding dipeptide or dipeptide ester. After aminoylsis, the ketoxime-derivatized resin can be reused. An advantageous solid-phase method is thus provided for making α-L-aspartyl dipeptide ester sweeteners, including aspartame, and the immunopotentiating dipeptide, α-L-glutamyl-L-asparagine.

This is a division of application Ser. No. 687,475, filed Dec. 28, 1984,now U.S. Pat. No. 4,600,532.

TECHNICAL FIELD

The present invention concerns processes and compositions for makingamino-terminal, α-aspartyl and α-glutamyl dipeptides and dipeptideesters. More particularly, the invention relates to solid phaseprocesses for making such peptides and peptide esters and to derivatizedpolymer resin intermediates in such processes.

BACKGROUND OF THE INVENTION

A number of α-L-aspartyl, dipeptide esters are known to be intenselysweet and useful as sweeteners. R. Mazur et al., J. Am. Chem. Soc. 91,2684-2691 (1969); U.S. Pat. Nos. 3,920,626; 3,492,131 and 3,475,403.Among these, aspartame (α-L-aspartyl-L-phenylalanine methyl ester) isnow being used extensively in the food and beverage industries.

α-L-glutamyl-L-asparagine, and pharmaceutically acceptable saltsthereof, are known to be therapeutically useful as immunopotentiatingagents in humans and other mammals. U.S. Pat. No. 4,426,324.

A number of methods of making aspartame and the other α-L-aspartyl,dipeptide ester sweeteners are also known. See, for example, thereferences cited above in connection with dipeptide ester sweeteners, aswell as Vinick and Jung, Tetrahedron Lett. 23, 1315-1318 (1982), andU.S. Pat. Nos. 3,962,207; 3,833,553; 3,798,206 and 3,786,039.

Several methods for making aspartame and other lower alkyl esters ofα-L-aspartyl phenylalanine have been disclosed wherein a strong acidsalt of L-aspartic anhydride is reacted with the methyl, or other loweralkyl, ester of L-phenylalanine or an acid solution thereof, see U.S.Pats. Nos. 3,962,207; 3,833,553; 3,798,206 and 3,786,039. A problemencountered in such syntheses is that the reactivity of the β-carboxylof the aspartic anhydride with the amino group of the phenylalanineester is approximately one quarter to the same as that of theα-carboxyl, unless the reaction is run under conditions which enhancethe reactivity of the α-carboxyl relative to that of the β-carboxyl. Theproduct resulting from condensation of the β-carboxyl, β-L-aspartylphenylalanine methyl ester, is bitter and, therefore, an undesirablecontaminant in aspartame preparations. It has been found that reactingstrong acid salts of L-aspartic anhydride with L-phenylalanine loweralkyl esters in the presence of weak acids reduces the reactivity of theβ-carboxyl of the aspartic anhydride, relative to that of theα-carboxyl, by as much as 50% and, further, increases the total yield ofboth of the dipeptide esters, see U.S. Pat. No. 3,833,553.

The use of oxime and ketoxime esters of covalently protected amino acidsin peptide synthesis is known. See, e.g., Hayashi and Shimizu, Bull.Chem. Soc. Japan 56, 3197-3198 (1983); Fujino and Nishimura, Chem.Pharm. Bull. 17, 1937 (1969). Aminolysis of oxime and ketoxime esters ofcovalently protected amino acids with amino acid esters, and catalysisof such aminolysis with weak acids, such as acetic acid or formic acid,are known. See, e.g., Hayashi and Shimizu, supra.

The use in solid-phase peptide synthesis of amino acid ketoximeester-derivatized polystyrene resins has been reported, wherein thepolymer-supported amino acid residues have protecting groups covalentlybound to their amino groups and the corresponding ketoxime is formedwith a phenyl group of the polystyrene support and a phenyl group, orsubstituted phenyl group selected from p-nitro, p-chloro and p-methoxyphenyl, provided by acylation of the polystyrene phenyl group with abenzoyl, or p-substituted benzoyl, halide. DeGrado and Kaiser, J. Org.Chem. 45, 1295-1300 (1980); DeGrado and Kaiser, J. Org. Chem. 47,3258-3261 (1982); Nakagawa and Kaiser, J. Org. Chem. 48, 678-685 (1983).Aminolysis with amino acid esters of the polymer-supported,p-nitrobenzophenone ketoxime esters of covalently protected amino acidsand peptides, and catalysis of such aminolysis with acetic acid, havealso been reported. DeGrado and Kaiser, 1980 and 1982, supra; Nakagawaand Kaiser, 1983, supra.

Ketoxime esters of aspartic acid and glutamic acid, without protectinggroups covalently bound to the amino group or unesterified carboxylgroup, have not been known heretofore.

SUMMARY OF THE INVENTION

It has now been discovered that a strong acid salt of aspartic orglutamic anhydride, without a covalently bound protecting group, reactswith ketoxime-derivatized polymer resins to form the correspondingnovel, polymer-supported ketoxime ester.

Further, it has been found that aminolysis in the presence of weak acidof the polymer-supported ketoxime ester, wherein the aspartyl orglutamyl group remains as the strong acid salt, with an amino acid esteror a salt of an amino acid with a base yields the correspondingdipeptide ester or dipeptide, respectively, and ketoxime-derivatizedresin. The ketoxime-derivatized resin can be reused.

The aspartic or glutamic anhydride strong acid salt reacts with theketoxime-derivatized resin predominantly through the α-carboxyl of theaspartyl or glutamyl group.

Further, in the aminolysis reaction, in the presence of weak acid, theamino acid ester or salt of an amino acid with a base will react, withnegligible racemization, predominantly at the polymer-bound carboxyl ofthe polymer-supported aspartyl or glutamyl group.

Thus, an advantageous, solid-phase method for synthesizing L-α-aspartyl,dipeptide ester sweeteners, especially aspartame, and L-α-glutomyldipeptides, such as L-α-glutamyl-L-asparagine, has been found. Among itsadvantages, the method involves no costly protection and deprotection ofamino or carboxyl groups.

The dipeptides and dipeptide esters made by the method of the inventionare useful per se or useful as intermediates in making polypeptides orpolypeptide esters which include the dipeptides or dipeptide esters intheir sequences.

DETAILED DESCRIPTION OF THE INVENTION

A process has been discovered for making a dipeptide or dipeptide ester,or a salt thereof, with an N-terminal α-aspartyl or α-glutamyl whichcomprises reacting a strong acid salt of aspartic anhydride or glutamicanhydride, respectively, with a ketoxima-derivatized polymer andreacting, in the presence of weak acid, the resulting novel aspartyl orglutamyl ketoxime ester-derivatized polymer, wherein the aspartyl orglutamyl groups remaing as the strong acid salt, with a salt with abase, or an ester, of the carboxy-terminal amino acid corresponding tothe dipeptide or dipeptide ester, respectively.

The invention thus entails a process for making a compound of formula I,or a salt thereof, ##STR1## wherein m is 1 or 2; wherein, when m is 2, nis 0, R₁ is --CH₂ C(O)NH₂, and R₂ is hydrogen;

wherein, when m is 1, n is 0, 1 or 2;

wherein, when m is 1 and n is 0, R₁ is benzyl, p-hydroxybenzyl,p-methoxybenzyl, p-ethoxybenzyl, methylthiomethyl, methylthioethyl,methylsulfonylethyl, or --CH₂ O(CO)R₃,

wherein R₃ is methyl, ethyl, n-propyl or i-propyl;

wherein, when n is 1 or 2, R₁ is hydrogen or methyl;

wherein, if R₁ is benzyl, p-hydroxybenzyl or methylthioethyl, R₂ ismethyl, ethyl, n-propyl, i-propyl or t-butyl;

wherein, if R₁ is hydrogen or methyl, R₂ is methyl, ethyl, n-propyl ori-propyl;

wherein, if R₁ is p-methoxybenzyl, p-ethoxybenzyl, methylthiomethyl,methylsulfonylethyl or --CH₂ O(CO)R₃, R₂ is methyl or ethyl;

wherein the configuration of the amino-terminal aspartyl or glutamylgroup is L; and

wherein the configuration at the carbon to which R₁ is bonded is L, if nis 0, and D, if n is 1 or 2 and R₁ is methyl, which comprises:

(i) reacting a strong acid salt of a compound of formula II ##STR2##wherein m is as in the compound of formula I and the configuration atthe asymmetric carbon is L or D,L, with a ketoxime-derivatized polymer;and

(ii) reacting, in the presence of weak acid, the novel aspartyl orglutamyl ketoxime ester-derivatized polymer from step (i), wherein theaspartyl or glutamyl group remains as a strong acid salt, with acompound of formula III ##STR3## wherein n and R₁ are as in the compoundof formula I; wherein, if R₂ in the compound of formula I is nothydrogen, R₄ is the same as R₂ ; wherein, if R₂ in the compound offormula I is hydrogen, R₄ is a cation corresponding to a base of whichthe compound of formula III is a salt; and wherein the configuration atthe asymmetric carbon is L or D,L, if n is 0, and D or D,L, if n is 1 or2 and R₁ is methyl.

Use herein of the term "ketoxime" indicates an oxime formed by reactionof hydroxylamine with a ketone.

Use herein of the terms "D,L" as a prefix to the name of a compound, ora designation of configuration at an asymmetric carbon in a compound,unless otherwise indicated, indicates a mixture of the enantiomorphs ofthe compound wherein each enantiomorph is a substantial fraction (i.e.,at least about 1%) of the mixture.

The compounds of formula I are known. Those wherein m is 1 are useful assweetners of foods and beverages. That wherein m is 2 is usefultherapeutically as an immunopotentiating agent in mammals, includinghumans (U.S. Pat. No. 4,426,324).

The preferred applications of the invention are to making the compoundsof formula I wherein m is 1, n is 0, R₁ is benzyl, p-hydroxybenzyl ormethylthioethyl, and R₂ is methyl, i.e. α-L-aspartyl-L-phenylalaninemethyl ester (also known as aspartame), α-L-aspartyl-L-tyrosine methylester, and α-L-aspartyl-L-methionine methyl ester. The most preferredapplication is to making aspartame.

Strong acid salts of L- and D,L-aspartic anhydride and L- andD,L-glutamic anhydride (compounds of formula II) are also known andinclude those of strong mineral acids, such a hydrochloric, hydrobromic,hydroiodic, sulfuric, chlorosulfonic, bromosulfonic, perchloric andnitric acids; those formed with monoesters of sulfuric acid, such amethylsulfuric, ethylsulfuric, isopropylsulfuric and benzylsulfuricacids; those formed with organic sulfonic acids, such asbenzenesulphonic, p-toluenesulfonic, β-naphthalenesulfonic, andalkylsufonic acids such as methylsulfonic, ethylsulfonic andisopropylsulfonic acid; and those formed with halogenated carboxylicacids, such as trichloroacetic, dichloroacetic and trifluoroaceticacids. See U.S. Pat. Nos. 3,462,460 and 3,816,471; and Ariyoshi et al.Bull. Chem. Soc. (Japan) 45, 2208 (1972) and 46, 2611 (1973). Theglutamic anhydride salts are prepared in essentially the same way as theaspartic anhydride salts, beginning with glutamic acid rather thanaspartic acid. Preferred for use in the present invention are thehydrochloric and methylsulfuric acid salts of aspartic and glutamicanhydride.

The compounds of formula III, wherein R₄ is the same as R₂ in thecorresponding compound of formula I, are known, both as essentially pureD or L isomer and as mixtures of both isomers.

R₄ in the compound of formula III, when R₂ in the corresponding compoundof formula I is hydrogen, can be any cation, corresponding to a base ofwhich the compound of formula III is a salt. The bases used to make thesalts of formula III are, generally, bases which are strong enough tomaintain the amino group on a substantial fraction of the amino acid inthe free amine form yet not so strong or nucleophilic as to causesignificant racemization of the amino acid. Such bases include sodium orpotassium bicarbonate, N-alkylmorpholines (e.g., N-methylomorpholine,N-ethylmorpholine), piperidine, and trialkylamines. Preferred aretrialkylamines wherein the alkyl moieties are the same or different andare each of 1 to 3 carbon atoms. Compounds of formula III which aresalts of amino acids with such bases are known, or readily synthesizedby the skilled, both as essentially pure D or L isomer and as mixturesof both isomers.

Weak acids which can be employed in the aminolysis reaction (reactionstep (ii), supra) with the compound of formula III include, generally,acids which, in aqueous solution at 25° C., have dissociation constantsless than 3×10⁻ M and preferably less than 10⁻⁴ M. If an acid has morethan one ionizable hydrogen atom, the dissociation constant for thefirst such hydrogen atom will be less than 3×10⁻² M, and preferably lessthan 10⁻⁴ M, in aqueous solution at 25° C. Such weak acids includephosphoric acid, phosphorous acid, carbonic acid, formic acid,alkylcarboxylic acids such as acetic, proprionic and n-butyric acids,monohalogenated alkylcarboxylic acids such as chloroacetic, bromoaceticand iodoacetic acids, citric acid, succinic acid and phenol. Preferredis acetic acid.

Preferably the amount of weak acid present in the aminolysis reactionmixture is equimolar with the total amount of compound of formula IIIinitially present in the reaction mixture. By "total amount of compoundof formula III" is meant the amount of such compound in both of itsforms: with the amino group protonated and as free amine. If R₂ in thecorresponding compound of formula I is hydrogen, the "total amount ofcompound of formula III" includes also the analogs of the compound, inboth protonated amine and free amine forms, wherein the carboxyl groupis in anionic form, protonated form, and associated with cations, otherthan R₄ and protons, that might be present.

Optionally, the addition salt, with the weak acid, of the compound offormula III can be used to provide both weak acid and compound offormula III to the aminolysis reaction mixture.

Ketoxime-derivatized polymers employed in the present invention includethose described by DeGrado and Kaiser, 1980 and 1982, supra.

Any polymer which can be ketone-derivatized can be employed to make theketozime-derivatized polymers utilized in the invention. Preferred ispolystyrene, optionally crosslinked by copolymerization with suitablecrosslinking compounds.

Most preferred for the present invention are polystyrene resinsoptionally crosslinked by co-polymerization with 0.5% to 3%, by weight,divinylbenzene. Preferably, the resin will be used in the form of small,spherical beads of about 100 to about 1000 mesh in size.

An example of resin beads that can be used to make ketoxime-derivatizedpolymer for use in the invention is Biobeads SX1™, a polystyrene-1%divinylbenzene resin provided in the form of spherical beads, 200-400mesh in size, by Bio-Rad Laboratories, Inc., Richmond, Calif., U.S.A.

To form the ketoxime-derivatized polymer for use in the invention, apolymer is typically first acylated at a repeating group to form theketone-derivatized polymer and the ketone-derivatized polymer is thenreacted with hydroxylamine to form the correspondingketoxime-derivatized polymer.

Ketozime-derivatized polymers for use in the invention include thosewherein the ketoxime-derivatized sites are represented by formula IV##STR4## wherein P represents the polymer backbone; the phenylene groupbetween P and C═N--OH is a repeating group in the polymer and is presentin the (polystyrene) polymer prior to formation of the ketoximederivative; wherein X₁₂ is (i) phenyl optionally substituted at any oneposition with chlorine; bromine; iodine; alkoxy, wherein the alkylmoiety is of 1 to 6 carbon atoms; sulfonyl; nitro; or trialkylamino,wherein the alkyl groups are the same or different and are each of 1 to4 carbon atoms; (ii) alkyl of 1 to 6 carbon atoms, optionallysubstituted at any one position with nitro, hydrogensulfate, sulfonyl,or trialkylamino, wherein the alkyl groups are the same or different andare each of 1 to 4 carbon atoms; (iii) cycloalkyl of 3 to 8 carbonatoms, optionally substituted at any one position with nitro,hydrogensulfate, sulfonyl, or trialkylamino, wherein the alkyl groupsare the same or different and are each of 1 to 4 carbon atoms; or (iv) aheterocyclic moiety consisting of 3 or 4 CH₂ groups in the ring; an atomor group selected from oxygen, nitrogen, sulfur and sulfonyl in thering; and a CH group in the ring and bonded to the ketoxime carbon atom;and wherein X₁₄ is a counterion to any charge that is present on X₁₂ andis the conjugate base of a strong acid. X₁₄ will typically be aunivalent anion corresponding to a strong acid, e.g., chloride ormethylsulfate.

Preferred among the polymers of formula IV are those wherein X₁₂ isphenyl or phenyl substituted in the para-position with methoxy,chlorine, bromine, nitro or (trimethyl)amino.

The preparation of ketoxime-derivatized polymers of formula IV is knownin the art. See DeGrado and Kaiser, 1980 and 1982, supra.

The process of the invention can also be carried out with aketoxime-derivatized polymer wherein the ketoxime-derivatized sites arerepresented by formula V ##STR5## wherein P represents the polymerbackbone; wherein the phenylene group between P and C═N--OH is arepeating group in the polymer and is present in the polymer prior toformation of the ketoxime derivative; wherein Z₁, Z₂ and Z₃ are at anythree positions on the phenyl group and are the same or different, eachbeing selected from hydrogen; chlorine; bromine; iodine; alkoxy,alkylsulfonyl, and alkylsulfonic, wherein the alkyl group is of 1 to 6carbon atoms; trialkylamino, wherein the alkyl groups are the same ordifferent and are each of 1 to 4 carbon atoms; nitro; and sulfonyl;provided that not more than one of Z₁, Z₂ and Z₃ is hydrogen; andwherein X₆ represents the counterion or counterions to any charge thatis present on the substituted phenyl group and consists of one or moreconjugate bases of strong acids. X₆ will typically be one or moreunivalent anions corresponding to strong acids, e.g. chloride,methylsulfate. Preferred among the polymers of formula V is that whereinthe substituted phenyl group is o-methoxy-p-(trimethylamino)phenyl.

The ketoxime-derivatized polymers according to formulas IV and V aremade in essentially the same way. First, the ketone-derivatized polymeris made by Friedel-Crafts acylation of the repeating phenyl groups ofthe polymer with a compound of formula VI or VII ##STR6## correspondingto the derivative to be added to the polymer. In formulas VI and VII, X₅is chlorine or bromine, preferably chlorine; X₁₂ and X₁₄ are as definedabove for the compound of formula IV; and Z₁, Z₂, Z₃ and X₆ are asdefined above for the compound of formula V. Compounds of formulas VIand VII are readily available or readily made by the skilled person.

The acylation of the resin is carried out in the presence of a Lewisacid catalyst, preferably AlCl₃, in an anhydrous, aprotic solvent suchas ethyl acetate, dimethylformamide, an aromatic or nitrated aromatichydrocarbon (e.g. benzene, toluene, nitrobenzene), a halogenatedhydrocarbon (e.g. 1,2-dicloroethane), a polyether (e.g. polyethyleneglycol), or a mixture of any of the foregoing.

As the skilled will recognize, the choice of solvent will be indicatedprimarily by the reactivity of the acylating compound. With the morereactive compounds, the reaction will be run at a temperature near orbelow room temperature (e.g. 0° C. to 30° C.); and any of the solventsor mixtures of solvents which remain sufficiently fluid at the reactiontemperature to permit efficient stirring can be used. With less reactiveacylating compounds, the reaction will need to be carried out at highertemperatures (e.g. 30° C. to 100° C.) to proceed reasonably rapidly; inthese cases, a solvent or mixture of solvents which refluxes at asuitable temperature is preferably used. The refluxing reaction mixturewill additionally be efficiently stirred.

Swelling of the resin that occurs with some of the solvents for theacylation reaction is also desirable.

The acylation reaction is carried out over about 4 to about 36 hours,preferably 8 to 24 hours, with a molar amount of acylating agent that istypically 5% to 50% of that of phenyl groups in the resin to beacylated.

The extent of acylation of the polymer will depend on a number offactors, including the reactivity of the acylating agent, theaccessibility of phenyl groups in the resin to acylating agent (which inturn will depend on the extent of swelling of the polymer, theconcentration of acylating agent in the reaction mixture, and theefficiency with which the stirring of the reaction mixture permitsacylating agent to diffuse into the polymer particles), and thetemperature and duration of the acylation reaction.

Compounds of formula VII, wherein one or more of Z₁, Z₂ and Z₃ would bereactive in the Friedel-Crafts acylation reaction mixture, must besuitably protected at these reactive groups prior to the acylationreaction and then deprotected after the acylation reaction.

The ketone-derivatized polymer is recovered by filtration and thoroughlywashed with suitable solvents to remove unreacted reagents. A suitablewashing protocol is provided in Example I.

Typically 5% to 30% of the phenyl groups of the polystyrene resin areacylated by the above procedure. The extent of acylation can bedetermined by a number of techniques known in the art, includingmeasuring weight-gain by the resin.

The ketoxime-derivatized polymer is then prepared as follows from thewashed, ketone-derivatized polymer:

The washed, ketone-derivatized polymer is added slowly, with efficientstirring, to a solution of a strong acid salt of hydroxylamine,approximately equimolar in base (e.g., pyridine), in a polar,non-aqueous solvent such as a lower alkanol or cyclic ether. Thesolution is efficiently stirred and maintained at 20° C.-120° C. forabout 4 to 48 hours. The preferred strong acid salt of the hydroxylamineis the hydrochloride, the preferred base is pyridine, the preferredsolvent is ethanol, the preferred reaction temperature is 80° C., andthe preferred reaction time is 8-12 hours. Hydroxylamine is present in5-50 fold molar excess, preferably 15-25 fold molar excess, with respectto ketone groups on the ketone-derivatized polymer.

"Efficient stirring" in the present specification means stirring orshaking that maintains the surface of resin particles in contact withsolution, so that reagent can readily diffuse into the particles toreach sites for reaction, but that is not so vigorous that it causesfragmentation of the resin particles.

Ketoxime-derivatized polymer is recovered from the reaction mixture byfiltration and is then thoroughly washed with suitable solvents toremove unreacted reagents and reaction by-products. A suitable washingprocedure is provided in the fourth paragraph of Example I.

After the washing, the resin is dried in vacuo.

The extent of conversion of ketone-carbonyl groups to ketoxime groups bythe above procedure is 90%-100%.

As indicated by the wavy line representing the bond between the ketoximehydroxyl, or ketoxime ester linked aspartyl or glutamyl group, andketoxime nitrogen in various figures in the present specificationrepresenting ketoxime-derivatized or ketoxime ester-derivatizedpolymers, such polymers utilized in the present invention have acombination of syn and anti configurations about the ketoxime C=N doublebond.

Novel, aspartyl or glutamyl ketoxime ester-derivatized polymer is thenformed from the ketoxime-derivatized polymer as follows:

To an efficiently stirred mixture of ketoxime-derivatized polymer in apolar, aprotic solvent, such as halogenated hydrocarbons, (e.g.,methylene chloride, chloroform), nitroalkanes (e.g., nitromethane),acetonitrile, cyclic ethers (e.g. tetrahydrofuran), dimethylformamide,ethylacetate, or a mixture of any of them, is added a strong acid saltof L- or D,L-aspartic anhydride or L- or D,L-glutamic anhydride. Theamount of anhydride salt added preferably approximately equals theamount required stoichiometrically for complete esterification ofketoxime groups in the resin.

After addition of the anhydride salt, the reaction is continued at atemperature, between about -50° C. to about 40° C., at which efficientstirring can be maintained, for from about 10 minutes to about 10 hours.Preferred solvents for the reaction are those which do not appreciablyshrink the ketoxime-derivatized resin and wherein the strong acid saltof glutamic or aspartic anhydride is sufficiently soluble for thederivativization reaction to proceed at a reasonably rapid rate.Non-aqueous, protic solvents (e.g. methanol) may be used but tend todestabilize the anhydrides. Generally, a solvent such as 50% (v/v)acetonitrile in tetrahydrofuran or 50% (v/v) ethylacetate in eitheracetonitrile or nitromethane will be acceptable. The preferredtemperature of the reaction is room temperature, i.e. approximately 20°C. to approximately 30° C. Typically, more than half of the ketoximenitrogens in the resin will be converted to ketoxime ester nitrogens inthe foregoing procedure.

After completion of the reaction, the aspartyl or glutamyl ketoximeester-derivatized polymer (resin) is separated by filtration and,preferably, thoroughly washed with a non-aqueous, polar, preferablyaprotic, solvent suitable to remove unreacted anhydride acid salt andreaction by-products. Nitromethane, acetonitrile, tetrahydrofuran,ethylacetate, dimethylformamide or mixtures of them are suitable forthis purpose.

The washing step after formation of the glutamyl or aspartyl ketoximeester-derivatized polymer is not necessary to obtain dipeptide ordipeptide ester product in accordance with the novel process describedherein. However, said washing step, by eliminating from the resinunreacted acid salt of aspartic or glutamic anhydride and various otherimpurities, substantially increases the yield of desired dipeptide ordipeptide ester product from the final step in the process, to bedescribed below.

The synthesis, and optional post-synthesis washing, of ketoxineester-derivatized resin are preferably carried out so that the aspartylor glutamyl residues on the resin remain as the strong acid salts.

As will be apparent to those of skill, if anions represented by X₁₄(Formula IV) or X₆ (Formula V) are present in the ketoxime-derivatizedpolymer, those anions and that corresponding to the strong acid of thestrong acid salt of the aspartyl or glutamyl group will intermix in theaspartyl or glutamyl ketoxime ester-derivatized resin. Thus, a fractionof the aspartyl or glutamyl groups in such resins will be associatedwith X₁₄ or X₆. X₁₄, or X₆, may be the same as, or different from, theanion corresponding to the strong acid of the strong acid salt ofaspartic or glutamic anhydride used to make the aspartyl or glutamylketoxime ester-derivatized resin.

In addition to the fact that the aspartyl and glutamyl ketoximeester-derivatized resin, when made as described above with a strong acidsalt of aspartic or glutamic anhydride, respectively, has no protectinggroups covalently bound to the aspartic acid or glutamic acid aminogroups or unesterified carboxyl groups, a preponderance of the aminoacid residues in the ketoxime ester-derivatized resin so formed areesterified through the α-carboxyl group.

Preparation of the compound of formula I from a compound of formula IIIand aspartyl or glutamyl ketoxime ester-derivatized polymer proceeds viaaminolysis of the ketoxime ester as follows:

A solution is prepared of a compound of Formula III, in a polar,non-aqueous solvent such as methanol, ethanol, tetrahydrofuran,acetonitrile, nitromethane, ethylacetate, dimethylformamide, or amixture of any of them.

To prepare the solution, the compound of Formula III itself can bedissolved to the desired concentration in the desired solvent.Alternatively, an acid addition salt (e.g. hydrochloride) of compound ofFormula III can be used to prepare the solution.

It is essential that a substantial fraction of the compound of formulaIII in this solution have the amino group in the free amine form. Thus,if an acid addition salt (e.g. hydrochloride) of a compound of formulaIII is used to prepare the solution, at least a portion of the acid ofthe acid addition salt must be neutralized. The base used in suchneutralization is strong enough so that, at the concentration employed,a substantial fraction of the compound of formula III will be in thefree amine form but not so strong or nucleophilic as to causesignificant racemization of the amino acid or amino acid ester. Suchbases include sodium or potassium bicarbonate, N-alkylmorpholines (e.g.N-methylmorpholine, N-ethylmorpholine), piperidine, and trialkylamines.Preferred are trialkylamines wherein the alkyl groups are the same ordifferent and are each of 1 to 3 carbon atoms. Preferably, the acidaddition salt will be neutralized with an equimolar amount of base inpreparing the solution of compound of Formula III.

In preparing a solution of a compound of Formula III, which correspondsto a compound of Formula I wherein R₂ is hydrogen, from an acideaddition salt of the amino acid corresponding to the compound of formulaIII, preferably the same base will be used to prepare the compound ofFormula III from the amino acid and neutralize the acid (e.g.hydrochloride) corresponding to the acid addition salt. If thecorresponding amino acid is used to prepare the compound of Formula III,preferably the amount of base used will be equimolar with the amount ofamino acid. If an acid addition salt of the amino acid is used toprepare the compound of Formula III, preferably the amount of base usedwill be twice the molar amount of the acid addition salt.

As indicated, preparation of a solution of a compound of Formula III caninvolve concomitant production of salts, other than compounds of FormulaIII, such as, e.g., trialkylammonium chlorides. By numerous methods wellknown in the art, (e.g. chromatographic, solvent extraction), thesesalts can optionally be separated from the compounds of Formula IIIbefore a solution of the compound of Formula III for use in theaminolysis reaction is finally prepared. If these salts are notseparated from the compound of Formula III prior to aminolysis, theywill be mixed with the compound of Formula I and salts thereof formed inthe aminolysis.

The aminolysis is conducted in the presence of weak acid. Thus, in thepreferred procedure, after the solution of compound of Formula III isprepared, it is mixed with an aliquot of weak acid and then used inaminolysis. The weak acid is preferably added directly to the solutionof compound of Formula III. It can, however, be added in solution in asuitable polar, non-aqueous solvent (e.g. methanol, ethanoltetrahydrofuran, acetonitrile, nitromethane, ethylacetate,dimethylformamide, or a mixture of any of them).

The amount of weak acid in the aminolysis reaction mixture is preferablyequimolar with the total amount of compound of Formula III initiallypresent in the reaction mixture. Thus, an addition salt of the weak acidwith a compound of Formula III may be used directly in preparing asolution to be used in aminolysis, rather than first preparing asolution of compound of formula III and then adding weak acid to thatsolution.

In yet another alternative procedure, rather than adding weak acid tothe solution of compound of Formula III to prepare a solution for theaminolysis, the weak acid can be added instead to the solvent in whichaspartyl or glutamyl ketoxime ester-derivatized resin is suspended forthe aminolysis, prior to addition of solution of compounds of FormulaIII to the resin suspension.

For the aminolysis, the solution of compound of Formula III, preferablyincluding an amount of weak acid equimolar with the total amount ofcompound of Formula III, is added to an efficiently stirred suspensionof aspartyl or glutamyl ketoxime ester-derivatized resin, wherein theaspartyl or glutamyl groups remain as strong acid salt, in a polar,non-aqueous solvent such as methanol, ethanol, tetrahydrofuran,acetonitrile, nitromethane, ethylacetate, dimethylformamide, or amixture of any of them. The solvent is preferably the same as that ofthe solution, of the compound of Formula III (preferably together withweak acid), added to the resin suspension for the aminolysis reaction.

The reaction mixture is stirred at 0° C. to 60° C., preferably at roomtemperature (20° C.-30° C.), for about 2 to 24 hours, preferably about 4to about 8 hours. The reaction can be run essentially to completion.

The total amount of compound of Formula III initially present in theaminolysis reaction mixture is 0.5 to 5 times, preferably 1 to 1.5times, the number of moles of the aspartyl or glutamyl groups bound inthe resin.

After completion of the aminolysis, resin is separated from solvent byfiltration and washed with polar, non-aqueous solvents suitable toremove reactants and reaction products from the resin. The washingprocedures in the final paragraph of Example I or in Examples IV or Vare suitable. Further, a suitable composition for the solvent used forwashing is the composition of the solvent in which the compound ofFormula III is dissolved for mixing with the suspension ofester-derivatized resin for the aminolysis reaction.

The washed resin is then recharged with strong acid salt of asparticanhydride or glutamic anhydride, as described above for formation of theaspartyl or glutamyl ketoxime ester-derivatized polymer, includingoptionally washing after the derivativization. The recharged resin isthen reused in an aminolysis reaction to make a compound of formula I.

It is found that a preponderance of the compound of formula III thatreacts, in the presence of weak acid, with the aspartyl or glutamylgroups in the ketoxime ester-derivatized resin, wherein the aspartyl orglutamyl groups are present as strong acid salt, reacts at theα-carboxyl of the aspartyl or glutamyl groups to form acid additionsalt(s) of the compound of formula I, if R₂ in said compound is nothydrogen, or acid addition salt(s) of compounds formed by replacing R₂in the compound of formula I, if R₂ is hydrogen, with R₄ (and othercations that might be present). The anion of the acid addition salt(s)will include the anion corresponding to the strong acid associated withthe aspartyl or glutamyl groups and X₁₄ (FIG. IV) or X₆ (FIG. V). If X₁₄(or X₆) differs from the anion of the strong acid salt of the aspartylor glutamyl groups, there will be different acid addition salts formed,one corresponding to said anion and another to X₁₄ (or X₆).

The filtrate obtained upon separation of resin from the aminolysisreaction mixture will include the weak acid, essentially completelyundissociated; the acid addition salt(s), described in the previousparagraph, of the compound of formula I, if R₄ is not hydrogen; or, ifR₂ is hydrogen, of salt(s) of the compound of formula I with hydrogenreplaced by R₄ and other cations that might be present; and salts ofbases, if base was used in preparing the solution of the compound ofFormula III used in aminolysis and salts (other than of compound ofFormula III) formed by said base were not separated from the solutionprior to its use in aminolysis; possibly unreacted compound of FormulaIII (as a salt or salts); and various other compounds (impurities).

The compound of Formula I, or a salt thereof, can be prepared from thisfiltrate, or from salts prepared from the filtrate, by methods,including acid-and base-neutralization, solvent extraction,recrystallization and chromatographic methods, well known in the art.See, e.g. U.S. Pat. Nos. 4,426,324; 3,920,626; 3,833,553 and 3,798,206;and Ariyoshi et al., Bull. Chem. Soc. (Japan) 46, 1898 (1973). Thecompound of Formula I, or a salt thereof, may be obtained in anhydrousor hydrated form.

The preferred salts of the compounds of formula I are those acceptablefor human consumption, for the compounds intended for use in foods andbeverages, or acceptable for administration to humans by injection, forthe compounds intended for therapeutic use. The preferred salts includethose formed with physiologically acceptable cations, such as sodium,potassium, calcium, magnesium or ammonium, as well as acid additionsalts, of the compound of formula I or a salt thereof prepared with oneor more physiologically acceptable cations, that are formed withphysiologically acceptable acids, such as acetic, hydrobromic,hydrochloric, and sulfuric.

If optically impure aspartic or glutamic anhydride or optically impurecompound of formula III is used in making compound of Formula I by theprocess of the present invention, the product from aminolysis will be amixture of stereoisomers, usually only one of which will be active (as asweetener or immunopotentiating agent). The presence of inactive isomerswith the active one will lessen, but not eliminate, the activity (i.e.sweetness or pharmacological activity) of the final product. Thus,resolution of the various isomers will not be necessary in manyapplications. If resolution of the isomers to isolate the active one isdesired, persons of skill in the art are aware of numerous methods forresolving them.

Persons of skill will also recognize how dipeptides and dipeptideesters, that can be made by the methods of the present invention, can beused as intermediates in the synthesis of polypeptides or polypeptideesters which include in their sequences the dipeptides and dipeptideesters provided by the present invention.

The present invention is illustrated further in the following examples:

EXAMPLE I Benzophenone Ketoxime-derivatized Resin

Benzophenone ketoxime-derivatized polymer (resin) is prepared followingessentially the procedure outlined in DeGrado and Kaiser, J. Org. Chem.47, 3258-3261 (1982).

1% divinylbenzene-polystyrene copolymer resin, in the form of beads witha 200-400 mesh diameter and sold as Biobeads SX1, is obtained fromBio-Rad Laboratories, Inc., Richmond, Calif., U.S.A.

9 g of benzoyl chloride and 12 g of AlCl₃ are dissolved in 100 ml ofnitrobenzene that has been dried over 3A molecular sieves. The resultingsolution is filtered into a dropping funnel.

70.0 g of the Biobeads SX1 resin beads is added to 1200 ml of1,2-dichloroethane, that has been distilled from phosphorous pentoxideimmediately prior to use; and to this mixture, which has been brought tovigorous reflux and is stirred efficiently (to maintain surface of resinbeads in contact with solvent and promote efficient transfer of reagentsinto the resin) in added dropwise, over a period of 30 minutes, theabove-described solution of benzoyl chloride and AlCl₃ in 100 ml ofnitrobenzene. Stirring and refluxing of the resulting mixture iscontinued for 12 hours; the resin is then filtered and washed asfollows: 3 times, each with 1 liter of 3 parts dioxane to 1 part 4N HCl;3 times, each with 1 liter of 3 parts dioxane to 1 part H₂ O; 3 times,each with 1 liter of dimethylformamide; and 3 times, each with 500 ml ofmethanol. Finally, the resin is dried in vacuo.

The resulting ketone polymer is added over a 30 minute period to anefficiently stirred, boiling solution of 70 g hydroxylaminehydrochloride, 100 ml pyridine and 500 ml ethanol. Efficient stirringduring addition of ketone polymer to the solution is essential forquantitative conversion of ketone to oxime. The resulting mixture isstirred under reflux for 8 hrs. after all ketone polymer has been added.Finally, the resin is collected by filtration and washed as follows: 3times, each with 500 ml of 3 parts methanol in 1 part water; 2 times,each with 500 ml of dimethylformamide; and 3 times, each with 300 ml ofmethanol.

EXAMPLE II p-Nitrobenzophenone Ketoxime-derivatized Resin

p-Nitrobenzophenone ketoxime-derivatized polymer (resin) was prepared inthe same way as the resin of Example I, except that 12 g ofp-nitrobenzoyl chloride was used in place of 9 g of benzoylchloride as astarting material. As determined by weight-gain of the resin, in theresulting ketone-derivatized polymer there were 0.59 mmol ofp-nitrobenzoyl added per gram of ketone-derivatized polymer. In theformation of ketoxime-drivatized polymer, essentially completeconversion of ketone to ketoxime occurred.

EXAMPLE III Phenyl, Methyl Ketoxime-derivatized Resin

To prepare the title resin, the procedure of Example I is used exceptthat 5 g of acetyl chloride is used in place of 9 g of benzoyl chlorideas starting material.

EXAMPLE IV Preparation of Aspartyl Ketoxime

Ester-derivatized Resin and α-L-Aspartyl-L-Phenylalanine Methyl Ester

13.0 g of p-nitrobenzophenone ketoxime-derivatized resin, that wasprepared substantially according to Example II and containedapproximately 10 mmole of ketoxime groups, was mixed with 150 ml of amixture of 2 parts (v/v) ethylacetate, 2 parts (v/v) acetonitrile and 1part (v/v) methanol (referred to hereafter in this Example as "2:2:1solvent"). To this resin mixture was added, with efficient stirring, 2.4g (10 mmole) of the methylsulfuric acid salt of L-aspartic anhydride.The stirring was continued for 6 hours at room temperature (approx. 25°C.), whereupon the derivativization of resin with aspartyl groupsappeared to be complete, as no aspartic anhydride salt was evident uponthin-layer chromatographic analysis of solvent.

The resulting aspartyl ketoxime ester-derivatized resin was washed 3times, each time with 75 ml of the 2:2:1 solvent.

2.1 g (10 mmole) of L-phenylalanine methyl ester hydrochloride wasdissolved in 75 ml of the 2:2:1 solvent. To this solution was firstadded 1.37 ml (10 mmole) triethylamine and, after 1-2 minutes of mixing,0.6 ml (10 mmole) of acetic acid. The resulting solution was then addedslowly to an efficiently stirred solution of the above-prepared aspartylketoxime ester-derivatized resin (comprising approximately 10 mmole ofaspartyl hydrogenmethylsulfate groups) in 75 ml of the 2:2:1 solvent.The reaction was continued with efficient stirring at room temperature(approx. 25° C.) for 24 hours. After 24 hours, resin was separated fromsolvent by filtration.

The filtrate was analyzed for products by high performance liquidchromatography (HPLC). Several products were observed. Dipeptide esterproducts eluted from the HPLC column as the perchloric acid salt. Oneproduct eluted in the same fraction as the perchloric acid salt ofgenuine aspartame had in a control run. One product, which elutedslightly more slowly than, and was present at about half the amount of,the aspartame salt, was identified as the corresponding salt ofβ-L-aspartyl-L-phenylalanine methylester.

EXAMPLE V Preparation of Glutamyl Ketoxime Ester-derivatized Resin andα-L-Glutamyl-L-Asparagine

17 g of benzophenone ketoxime-derivatized resin according to Example I(and containing approximately 10 mmole of ketoxime groups) is mixed with150 ml of 1 part (v/v) tetrahydrofuran and 1 part (v/v) acetonitrile(referred to hereafter as "1:1 solvent"). To this resin mixture isadded, with efficient stirring, 1.7 g (10 mmole) of L-glutamic anhydridehydrochloride. The stirring is continued for 6 hours at room temperature(25° C.).

The resulting glutamyl ketoxime ester-derivatized resin is washed asfollows: 3 times, each with 75 ml of 1:1 solvent; 3 times, each with 50ml of acetonitrile; and, finally, 2 times, each 75 ml of 1:1 solvent.

2.0 g (12 mmole) of L-asparagine hydrochloride is dissolved in 1000 mlof 1:1 solvent at room temperature. To this solution, maintained at roomtemperature, is added, with efficient stirring, 3.5 ml (20 mmole) ofdiisopropylethylamine (DIEA).

The washed glutamyl ketoxime ester-derivatized resin, comprisingapproximately 10 mmole of glutamyl hydrochloride groups, is mixed with200 ml of 1:1 solvent and to the mixture is added 0.6 ml (10 mmole) ofacetic acid. The mixture is then stirred efficiently while theabove-described solution of L-asparagine hydrochloride with DIEA isadded to it. The reaction is continued at room temperature withefficient stirring for 24 hours. After 48 hours, the resin is separatedfrom solvent by filtration.

The filtrate includes the diisopropylethylammonium salt ofα-L-glutamyl-L-asparagine hydrochloride.

The foregoing example illustrate the present invention, but are notintended to limit the scope of the invention. Those skilled in the artwill recognize modifications and variations of the exemplifiedembodiments that are within the spirit and scope of the inventiondescribed and claimed in the present specification.

What is claimed is:
 1. A ketoxime ester-derivatized resin wherein theester-derivatized sites are represented by formula X ##STR7## wherein Prepresents the polymer backbone of the resin; wherein the phenylenegroup is a repeating functional group in the corresponding underivatizedpolymer; wherein X₁₁ is a strong acid salt of L- or D,L-aspartyl or astrong acid salt of L- or D,L-glutamyl, which is bonded to theketoxime-nitrogen through the α or β carboxyl group, and is notcovalently protected at either the amino group or the carboxyl groupthat is not bonded to the ketoxime-nitrogen, provided that the fractionof X₁₁ groups in the resin bonded to the ketoxime-nitrogen through theα-carboxyl and with a configuration of L is greater than 0; wherein X₁₂is (i) phenyl, or phenyl substituted at any one position with chlorine;bromine; iodine; alkoxy, wherein the alkyl moiety is of 1 to 6 carbonatoms; sulfonyl; nitro; or trialkylamino, wherein the alkyl groups arethe same or different and are each of 1 to 4 carbon; (iii) cycloakyl of3 to 8 carbon atoms; or (iv) a heterocyclic moiety consisting of 3 or 4carbon atoms in the ring, an atom or group selected from oxygen,nitrogen, sulfur and sulfonyl in the ring, and a CH group in the ringand bonded to the ketoxime carbon atom; and wherein X₁₃ is a counterionto any positive charge that is present on X₁₂ and is the conjugate baseof a strong acid.
 2. A ketoxime ester-derivatized resin according toclaim 1 wherein P is polystyrene optionally cross-linked with 0.5% to3%, by weight, divinylbenzene; X₁₁ is a strong acid salt of L-aspartylor a strong acid salt of L-glutamyl; and X₁₂ is methyl, phenyl,p-nitrophenyl, p-chlorophenyl, p-bromophenyl, p-methoxyphenyl, orp-(trimethylamino)phenyl; provided that, if X₁₂ is(trimethylamino)phenyl, at least a portion of the counterionsrepresented by X₁₃ is chloride or bromide.
 3. A ketoximeester-derivatized resin according to claim 2 wherein X₁₁ is L-aspartylchloride, bromide, iodide, methylsulfate, ethylsulfate,isopropylsulfate, benzylsulfate, methylsulfonate, ethylsulfonate,benzenesulfonate, p-toluenesulfonate, β-naphthalensulfonate,chlorosulfonate, bromosulfonate, nitrate, perchlorate, trichloroacetate,trifluoroacetate or dichloroacetate; provided that, if X₁₂ is(trimethylamino)phenyl, at least a portion of the X₁₁ groups isL-aspartyl hydrochloride or L-aspartyl hydrobromide.
 4. A ketoximeester-derivatized resin according to claim 2 wherein X₁₁ is L-glutamylchloride, bromide, iodide, methylsulfate, ethylsulfate,isopropylsulfate, benzylsulfate, methylsulfonate, ethylsulfonate,benzenesulfonate, p-toluenesulfonate, β-naphthalensulfonate,chlorosulfonate, bromosulfonate; nitrate; perchlorate; trichloroacetate,trifluosoacetate or dichloroacetate; provided that, if X₁₂ is(trimethylamino)phenyl, at least a portion of the X₁₁ groups isL-glutamyl hydrochloride or L-glutamyl hydrobromide.
 5. A ketoximeester-derivatized resin according to claim 3 wherein X₁₂ is phenyl orp-nitrophenyl and X₁₁ is L-aspartyl hydrochloride or L-aspartylhydrogenmethylsulfate.
 6. A ketoxime ester-derivatized polymer accordingto claim 4 wherein X₁₂ is phenyl or p-nitrophenyl and X₁₁ is L-glutamylhydrochloride or L-glutamyl hydrogenmethylsulfate.